Integrin antagonists

ABSTRACT

Novel compounds, their salts and compositions related thereto having activity against mammalian integrins are disclosed. The compounds are useful in vitro or in vivo for preventing or treating thrombotic or restenotic disorders.

RELATED APPLICATION DATA

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/328,197, filed Jun. 8, 1999 which is a continuation ofinternational application number PCT/US97/22495 filed Dec. 8, 1997 underthe Patent Cooperation Treaty, which is a continuation-in-part of U.S.patent application Ser. No. 08/762,117 filed Dec. 9, 1996 which wasconverted to provisional application Ser. No. 60/040,063 on Feb. 27,1997.

FIELD OF THE INVENTION

[0002] This invention relates to novel α-sulfonamido and α-sulfinamidocontaining carboxylic acid compounds which are potent inhibitors ofArginyl-Glycyl-Aspartyl-(RGD)-dependent integrins. In another aspect,the present invention relates to these α-sulfonamido and α-sulfinamidocontaining carboxylic acids, their pharmaceutically-acceptable salts,and pharmaceutically-acceptable compositions thereof which are useful aspotent inhibitors of integrin adhesive functions in mammals. In yetanother aspect, the invention relates to methods for using theseinhibitors as therapeutic agents for disease states in mammalscharacterized by aberrant cellular adhesive disorders that occur duringthrombosis and restenosis.

BACKGROUND OF THE INVENTION

[0003] Cellular adhesion is believed to play an important role in boththrombus formation and cellular responses to vascular injury, as well asfor normal hemostasis. Vascular injury and thrombosis are prevalentduring the development and progression of vascular disease states. Theseinclude conditions such as atherosclerosis, acute myocardial infarction,chronic stable angina, unstable angina, transient ischemic attacks,stroke, peripheral vascular disease, arterial thrombosis, and conditionsinduced by interventional procedures such as restenosis followingangioplasty.

[0004] Cellular adhesions can be characterized as either cell-celladhesions or cell-matrix adhesions. Cells utilize a variety of cellsurface adhesion receptors and adhesive proteins to facilitate theseadhesive interactions. For cell-cell type adhesions, platelets play amajor role in this type of adhesive interaction that occurs during acutethrombosis. Platelet aggregation, thrombus formation and consolidationof clots mediated by platelets are principally achieved by adhesiveprotein crosslinking of the platelet glycoprotein (GPIIb-IIIa) alsoreferred to as α_(IIβ)β₃ which is found on the platelet surface. Thisheterodimeric adhesion receptor is one member of a large family ofheterodimeric transmembrane glycoprotein receptors, called integrins(Hynes, R. O., “Integrins: Versatility, Modulation and Signaling in CellAdhesion”, Cell 69:11 (1992)).

[0005] Other integrins which may have important cell adhesion functionsin thrombosis, hemostasis or in disease states characterized by vascularinjury are the vitronectin receptors (α_(v)β₃ and α_(v)β₅) and thefibronectin receptor (α₅β₁) In particular, the vitronectin receptor,α_(v)β₃, has been postulated to play roles in cellular migration ofsmooth muscle cells following vascular injury that can ultimately leadto restenosis of the vessel (Yue, T. L., et at., “Osteopontin-StimulatedVascular Smooth Muscle Cell Migration is Mediated by β₃ Integrin”, Exp.Cell. Res. 214:459-464 (1994); Choi, E. T., et al., “Inhibition ofNeointimal Hyperplasia by Blocking α_(v)β₃ Integrin with a Small PeptideAntagonist GpenGRGDSPCA”, J. Vasc. Surg. 19:125-134 (1994); Matsuno, H.,“Inhibition of Integrin Function by A Cyclic RGD-Containing PeptidePrevents Neointima Formation”, Circulation 90:2203-2206 (1994)). Anumber of the natural ligands of these integrins (e.g. α_(IIβ)β₃,α_(v)β₃, α_(v)β₅, and α₅β₁) such as fibrinogen, fibronectin, vonWillebrand factor, thrombospondin, osteopontin, vitronectin and others,contain and utilize the tripeptide sequence, Arg-Gly-Asp (RGD) to bindto their respective integrins. Small synthetic peptides containing theRGD sequence have been shown to bind to these integrins and to competefor the binding of natural adhesive ligands (Rouslahti, E. andPierschbacher, M. D.,“New Perspectives in Cell Adhesion: RGD andIntegrins”, Science 238:491-497 (1987)). Peptides containing the RGDsequence or mimetic compounds have thus been the basis for the discoveryof several potent and highly specific inhibitors of platelet α_(IIβ)β₃which are useful as antithrombotic agents. This literature has beenextensively reviewed. See Coller, B. S., “Blockade of PlateletGPIIb/IIIa Receptors as an Antithrombotic Strategy”, Circulation92:2373-2380 (1995); Cook, N. S., et al., “Platelet GlycoproteinIIb/IIIa Antagonists”, Drugs of the Future 19:135-159 (1994); T.Weller., et al., “Fibrinogen Receptor Antagonists—A Novel Class ofPromising Antithrombotics”, Drugs of the Future 19:461 (1994); andZablocki, J. A., et al., “Fibrinogen Receptor Antagonists”, Exp. Opin.Invest. Drugs, 3:437-448 (1994).

[0006] Highly specific inhibitors of α_(v)β₃ based on the RGDrecognition sequence have also been recently described. Specifically,the cyclic peptide, cyclo(Arg-Gly-Asp-D-Phe-Val] is a very potent andspecific inhibitor of the vitronectin receptor α_(v)β₃ (Pfaff, M., etal., “Selective Recognition of Cyclic RGD Peptides of NMR DefinedConformation by α_(IIβ)β₃, and α₅β₁ Integrins”, J. Biol. Chem.269:20233-20238 (1994); Jonczyk, A., et al., European Patent Application578083A2 (1994)).

[0007] The present invention describes the preparation of novelcompounds which inhibit the adhesive function of various RGD-dependentintegrins. More specifically, the novel compounds are non-specificinhibitors of the platelet integrin α_(IIβ)β₃ and the vitronectinreceptor α_(v)β₃.

SUMMARY OF THE INVENTION

[0008] The present invention relates to novel α-sulfonamido andα-sulfinamido containing carboxylic acids or carboxylic esters, theirpharmaceutically-acceptable .stereoisomers, salts, hydrates, solvatesand prodrug derivatives, and pharmaceutically-acceptable compositionsthereof which have particular biological properties and are useful aspotent antithrombotics and/or antirestenotic agents in mammals.

[0009] The present invention provides a compound of the formula:

[0010] wherein:

[0011] Y is selected from the group consisting of —COOH, —PO₃H₂, —SO₃Hand —COOR⁴; where R⁴ is selected from the group consisting ofC₁₋₁₀alkyl, C₁₋₈alkylaryl, aryl-C₁₋₈alkyl,C₁₋₈alkyloxycarbonyloxy-C₁₋₈alkyl, aryloxycarbonyloxy-C₁₋₈alkyl,

[0012] C₁₋₈alkyloxycarbonyloxyaryl, C₁₋₈alkylcarbonyloxy-C₁₋₈alkyl,arylcarbonyloxy-C₁₋₈alkyl and C₁₋₈alkylcarbonyloxyaryl;

[0013] A is selected from the group consisting of C₆₋₁₂alkyl,C₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl, C₀₋₈-alkyl-CO—NR⁵—CO_(0-t8)alkyl,C₀₋₈alkyl-O—C₀₋₈-alkyl, C₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl-NR⁵-CO—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—CO—C₁₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-CO—NR⁵-C₁₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl-CO—NR⁵—CO-alkyl,Cll₈alkyl-CO—C₁₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-CO—C₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-O—C₂₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-O—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-O—C₂₋₈alkyl-O—C₀₋₈alkyl-CO—NR⁵-CO-alkyl,C₀₋₈alkyl-S—C₀₋₈alkyl, C₀₋₈alkyl-S(O_(n))-C₀₋₈alkyl,C₀₋₈alkyl-S-C₂₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-S(O_(n))-C₂₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-S-C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-S(O_(n))-C₁₋₈-alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl-S—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl-S(O_(n))-C₁₋₈alkyl,C₀₋₈alkyl-CO—NR⁵-C₂₋₈alkyl-S-C₀₋₈-alkyl,C₀₋₈alkyl-CO—NR⁵-C₂₋₈alkyl-S(O_(n))-C₀₋₈alkyl,C₀₋₈alkyl-NR⁵-C₀₋₈alkyl-CO₂—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵-C₀₋₈alkyl-CS-O—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵-C₀₋₈alkyl-CO—NR⁵-C₀₋₈alkyl,C₀₋₈alkyl-NR⁵-C₀₋₈alkyl-CS-NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-O—C₀₋₈alkyl-CO₂—C₀₋₈alkyl,C₀₋₈alkyl-O—C₀₋₈alkyl-CS-O—C₀₋₈alkyl; C₀₋₈alkyl-SiR⁷R⁸-alkyl,C₀₋₈alkyl-SiR⁷R⁸—C₀₋₈alkyl-NR⁶—CO—C₀₋₈alkyl, andC₀₋₈alkyl-SiR⁷R⁸-C₀₋₈alkyl-CO—NR—CO-alkyl; where R⁵, R⁶, R⁷ and R⁸ areindependently selected from the group consisting of H and C ₁₋₆alkyl;and where n=1 or 2;

[0014] Z is selected from the group consisting of —NH—C(NR⁹R¹⁰)═NR¹¹,—NH—C(R⁹)═NR¹¹, —C(NR⁹R¹⁰)═NR and piperidinyl; where R⁹, R¹⁰ and R¹¹ areindependently selected from the group consisting of H, C₁₋₆alkyl,aryl-C₁₋₃alkyl and aryl; or where two of the R⁹, R¹⁰ or R¹¹ substituentsform a cyclic ring containing (CH₂)_(p), where p=2-5;

[0015] R¹ is H;

[0016] R² is selected from the group consisting of —SO_(m)-aryl,—SO_(m)—C₁₋₁₀alkyl and —SO_(m)-heteroaryl, where m=1 -2;

[0017] R³ is selected from the group consisting of H, C₁₋₈alkyl, aryl,C₁₋₈alkylaryl and heteroaryl;

[0018] and all pharmaceutically-acceptable stereoisomers, salts,hydrates, solvates and prodrug derivatives thereof.

[0019] In certain aspects of this invention, compounds are providedwhich are useful as diagnostic reagents. In another aspect, the presentinvention includes pharmaceutical compositions comprising apharmaceutically-effective amount of the compounds of this invention anda pharmaceutically-acceptable carrier. In yet another aspect, thepresent invention includes methods which comprise administering thecompounds of the present invention and pharmaceutical compositionsthereof for preventing or treating disease states characterized bythrombosis or vascular injury in mammals. Optionally, the methods ofthis invention comprise administering such pharmaceutical compositionsin combination with an additional therapeutic agent such as anantithrombotic, a thrombolytic agent or an anticoagulant, or anycombination thereof.

[0020] The preferred compounds also include theirpharmaceutically-acceptable stereoisomers, hydrates, solvates, salts andprodrug derivatives.

DETAILED DE4SCRIPTION OF THE INVENTION Definitions

[0021] In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unlessexplicitly stated otherwise.

[0022] The term “alkyl” refers to saturated and unsaturated aliphaticgroups including straight-chain and branched-chain and cyclic groups, orany combination thereof, having the number of carbon atoms specified, orif no number is specified, having up to 12 carbon atoms. Cyclic alkylstypically comprise a monocyclic aliphatic ring having 3 to 12 carbonatoms and preferably 3 to 7 carbon atoms. The cyclic alkyls of thisinvention may include one or more nitrogen atoms. Preferably, “alkyl”refers to straight-chain and branched-chain groups; more preferablystraight-chain groups.

[0023] The term “aryl” refers to an unsubstituted or substitutedaromatic ring, substituted with one, two or three substituents selectedfrom C₁₋₆alkoxy, C₁₋₆alkyl, C₁₋₆alkylamino, hydroxy, halogen, cyano,hydroxyl, mercapto, nitro, thioalkoxy, carboxaldehyde, carboxyl,carboalkoxy and carboxamide, including but not limited to carbocyclicaryl, heterocyclic aryl, and biaryl groups and the like, all of whichmay be optionally substituted. Preferred aryl groups include phenyl,halophenyl, C₁₋₆alkylphenyl, naphthyl, biphenyl, phenanthrenyl,naphthacenyl, and aromatic heterocyclics. The term “heteroaryl” as usedherein refers to any aryl group, containing from one to fourheteroatoms, selected from the group consisting of nitrogen, oxygen andsulfur.

[0024] The term “arylalkyl” refers to one, two, or three aryl groupsappended to an alkyl group having the number of carbon atoms designated.Suitable arylalkyl groups include, but are not limited to, benzyl,picolyl, naphthylmethyl, phenethyl, benzhydryl, trityl, and the like,all of which may be optionally substituted. Similarly, the term“alkylaryl” refers to an alkyl group, having the number of carbon atomsdesignated, appended to one, two, or three aryl groups.

[0025] The terms “halo” or “halogen” as used herein refer to Cl, Br, For I substituents.

[0026] The term “oxy” refers to an oxygen (O) atom. The terms “alkyloxy”and “aryloxy” thus refer to the respective groups positioned adjacent toan oxygen atom. The term carbonyloxy refers to —C(O)—O—.

[0027] The term “pharmaceutically-acceptable salts” includes salts ofcompounds derived from the combination of a compound of the presentinvention and an organic or inorganic acid. These compounds are usefulin both free base and salt form. In practice, the use of the salt formamounts to use of the base form; both acid and base addition salts arewithin the scope of the present invention.

[0028] “Pharmaceutically-acceptable acid addition salt” refers to thosesalts which retain the biological effectiveness and properties of thefree bases and which are not biologically or otherwise undesirable,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,hyroiodic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and organic acids such as acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like.

[0029] “Pharmaceutically-acceptable base addition salts” include thosederived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Particularly preferred are the ammonium, potassium,sodium, calcium and magnesium salts. Salts derived frompharmaceutically-acceptable organic nontoxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basicion-exchange resins, such as isopropylamine, trimethylamine,diethylamine, triethylamine, tripropylamine, ethano,amine,2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine,arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purines,piperizine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic nontoxic bases are isopropylamine,diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline,and caffeine.

[0030] The term “prodrug derivatives” refers to compounds of theinvention which have metabolically cleavable groups and become, bysovolysis or under physiological conditions, compounds of the inventionwhich are pharmaceutically-active in vivo. Fro example, esterderivatives of compounds of this invention are often active in vivo, butmay have only weak or no activity in vitro. Other derivatives of thecompounds of this invention have activity in both their acid and acidderivative forms, but the acid derivative form often offers advantagesof solubility, tissue compatibility, or delayed release in the mammalianorganism. See Bundgard, H., “Design of Prodrugs”, pp. 7-9, 21-24,Elsevier, Amsterdam, 1985. Prodrugs include acid derivatives well knownto practitioners of the art, such as esters prepared by the reaction ofthe parent acid with a suitable alcohol, or amides prepared by reactionof the parent acid with an amine. Simple aliphatic or aromatic estersderived from acidic groups pendant on the compounds of this inventionare preferred prodrug derivatives. In some cases, it is desirable toprepare double ester-type prodrugs such as (aclyoxy)alkyl esters or[(alkoxycarbonyl)oxy]alkyl esters.

[0031] “Biological property” for the purposes herein means an in vivoeffector or antigenic function or activity that is directly orindirectly performed by a compound of this invention. Effector functionsinclude receptor or ligand binding, any enzyme activity or enzymemodulatory activity, any carrier binding activity, any hormonalactivity, any activity in promoting or inhibiting adhesion of cells toan extracellular matrix or cell surface molecules, or any structuralrole. Antigenic functions include possession of an epitope or antigenicsite that is capable of reacting with antibodies raised against it.

[0032] In addition, the following are used in this application:

[0033] “Boc” t-butoxycarbonyl

[0034] “BOP” benzotriazol-1-yloxy-tris-(dimethylamino)phosphoniumhexafluorophosphate

[0035] “Cbz” benzyloxycarbonyl

[0036] “DCC” N,N′-dicyclohexylcarbodiimide

[0037] “DIEA” diisopropylethylamine

[0038] “DMAP” 4-dimethylaminopyridine

[0039] “DMF” N,N-dimethylformamide

[0040] “HBTU” 2-(1-H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphate

[0041] “HF” hydrogen fluoride

[0042] “HOBt” N-hydroxybenzotriazole

[0043] “MeOH” methanol

[0044] “Ph” phenyl

[0045] “PhSO₂NH—” phenylsulfonamido

[0046] “TFA” trifluoroacetic acid

[0047] In the compounds of this invention, carbon atoms bonded to fournon-identical substituents are asymmetric. Accordingly, the compoundsmay exist as stereoisomers, including enantiomers and diastereomers. Thecompounds of this invention having one or more centers of asymmetry mayexist as enantiomers or mixtures thereof (e.g. racemates). In addition,compounds that have two or more asymmetric centers can exist asdiastereomers. The syntheses described herein may employ racemates,enantiomers or diastereomers as starting materials or intermediates.Diastereomeric products resulting from such syntheses may be separatedby chromatographic or crystallization methods, or by other methods knownin the art. Likewise, enantiomeric product mixtures may be separatedusing methods known in the art. See for example, Jacqes, Collet andWilen “Enantiomers, Racemates and Resolutions” (Krieger Publishing Co.,Malabar, Fla. 1991). Each of the asymmetric centers, when present in thecompounds of this invention, may be in one of two configurations (R orS), and both are within the scope of the present invention. Some of thecompounds may be designated either D or L, which is a less preferredindicator of the configuration of the compound, based on the compound ofthis invention having a configuration that is similar to known aminoacids. In the processes described above, the final products may, in somecases, contain a small amount of the products having D or L-formresidues; however, these products do not affect their therapeutic ordiagnostic application.

[0048] In all of the compounds of the invention having one or more amidelinkages (—CO—NH—), such amide linkages may optionally be replaced withanother linkage which is an isostere such as —CH₂NH—, —CH₂S—, CH₂—O,CH₂CH₂, —CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, —CH₂SO—, andCH₂SO₂. This replacement can be made by methods known in the art. Thefollowing references describe preparation of peptide analogs whichinclude these alternative-linking moieties: Spatola, A. F., Vega Data(March 1983), Vol. 1, Issue 3, “Peptide Backbone Modifications” (generalreview); Spatola, A. F., in “Chemistry and Biochemistry of Amino Acids,Peptides and Proteins,” B. Weinstein, eds., Marcel Dekker, New York, p.267 (1983) (general review); Morley, J. S., Trends Pharm Sci pp. 463-468(1980) (general review); Hudson, D., et al., Int J Pept Prot Res14:177-185 (1979) (—CH₂NH—, —CH₂CH₂—); Spatola, A. F., et al., Life Sci38:1243-1249(1986) (—CH₂—S); Hann, M. M., J Chem Soc Perkin Trans I pp.307-314 (1982) (—CH═CH—, cis and trans); Almquist, R. G., et al., J MedChem 23:1392-1398 (1980) (—COCH₂—); Jennings-White, C., et al.,Tetrahedron Lett 23:2533(1982) (—COCH₂—); Szelke, M., et al., EuropeanApplication EP 45665; CA 97:39405 (1982) (—CH(OH)CH₂—); Holladay, M. W.,et al., Tetrahedron Lett 24:4401-4404 (1983) (—C(OH)CH₂—); and Hruby, V.J., Life Sci 31:189-199 (1982) (—CH₂—S—).

Preferred Embodiments

[0049] In preferred embodiments, the present invention providescompounds of the formula:

[0050] wherein:

[0051] Y is selected from the group consisting of —COOH, —PO₃H₂, —SO₃Hand —COOR⁴; where R⁴ is selected from the group consisting ofC₁₋₁₀alkyl, C₁₋₈alkylaryl, aryl-C₁₋₈alkyl,C₁₋₈alkyloxycarbonyloxy-C₁₋₈alkyl, aryloxycarbonyloxy-C₁₋₈alkyl,C₁₋₈alkyloxycarbonyl-oxyaryl, C₁₋₈alkylcarbonyloxy-C₁₋₈alkyl,arylcarbonyloxy-C₁₋₈alkyl and C₁₋₈alkylcarbonyl-oxyaryl;

[0052] A is selected from the group consisting of C₆₋₁₂alkyl,C₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl, C₀₋₈alkyl-CO—NR⁵-C₀₋₈alkyl,C₀₋₈alkyl-O—C₀₋₈alkyl, C₀₋₈alkyl-NR⁵—CO—C₁₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—CO—C₁₋₈alkyl-CO—NR⁵—C₀₋₈-alkyl,C₀₋₈alkyl-CO—NR⁵-C₁₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-CO—C₁₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-CO—C₀₋₈alkyl-NR⁵—CO—C₀₋₈-alkyl,C₀₋₈alkyl-O—C₂₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-O—C₀₋₈alkyl-CO—NR⁵-C₀₋₈alkyl,C₀₋₈alkyl-O—C₂₋₈alkyl-O—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-S—C₀₋₈alkyl, C₀₋₈alkyl-S(O_(n))—C₀₋₈alkyl,C₀₋₈alkyl-S—C₂₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-S(O_(n))—C₂₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-S—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-S(O_(n))—C₁₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl-S—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—CO—C₁₋₈alkyl-S(O_(n))—C₀₋₈alkyl,C₀₋₈alkyl-CO—NR⁵-C₂₋₈alkyl-S—C₀₋₈alkyl,C₀₋₈alkyl-CO—NR-C₂₋₈alkyl-S(O_(n))-C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—C₀₋₈alkyl-CO₂-C₀₋₈alkyl,C₀₋₈alkyl-NR⁵-C₀₋₈alkyl-CS—O—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵-C₀₋₈alkyl-CO—NR⁵-C₀₋₈alkyl,C₀₋₈alkyl-NR⁵-C₀₋₈alkyl-CS—NR⁵-C₀₋₈alkyl,C₀₋₈alkyl-O—C₀₋₈alkyl-CO₂-C₀₋₈alkyl,C₀₋₈alkyl-O—C₀₋₈-alkyl-CS—O—C₀₋₈-alkyl; C₀₋₈alkyl-SiR⁷R⁸-C₀₋₈alkyl,C₀₋₈alkyl-SiR⁷R⁸-C₀₋₈alkyl-NR⁶—CO—C₀₋₈alkyl, andC₀₋₈alkyl-SiR⁷R⁸-C₀₋₈alkyl-CO—NR⁶-C₀₋₈alkyl; where R⁵, R⁶, R⁷ and R⁸ areindependently selected from the group consisting of H and C₁₋₆alkyl; andwhere n=1 or 2;

[0053] Z is selected from the group consisting of —NH—C(NR⁹R⁰)═NR,—NH—C(R⁹)═NR, —C(NR⁹R¹⁰)═NR¹¹ and piperidinyl; where R⁹, R¹⁰ and R¹¹ areindependently selected from the group consisting of H, C₁₋₆alkyl,aryl-C₁₋₃alkyl and aryl; or where two of the R⁹, R¹⁰ or R¹¹ substituentsform a cyclic ring containing (CH₂)_(p), where p=2-5;

[0054] R¹ is H;

[0055] R² is selected from the group consisting of —SO_(m)-aryl,—SO_(m)-C₁₋₁₀alkyl and —SO_(m)-heteroaryl, where m=1-2;

[0056] R³ is selected from the group consisting of H, C₁₋₈alkyl, aryl,C₁₋₈alkylaryl and heteroaryl;

[0057] and all pharmaceutically-acceptable stereoisomers, salts,hydrates, solvates and prodrug derivatives thereof.

[0058] It is understood that the “A” substituents can be incorporated inthe compounds of the invention in the order written above or in thereverse order. For example, a suitable “A” substituent isC₀₋₈alkyl-O—C₀₋₈alkyl-CO—NR⁵-C₀₋₈alkyl. It is understood that the “Z”substituent can be positioned to the right or to the left of thissequence.

[0059] Preferred “Y” substituents are —COOH and —COOR⁴, more preferably—COOH. R⁴ is preferably C₁₋₁₀alkyl.

[0060] Preferred “A” substituents are selected from the group consistingof C₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl, C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₁₋₈alkyl-NR⁵—CO—C₁₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl-CO—NR⁵-C₀₋₈alkyl,C₁₋₈alkyl-CO—NR⁵-C₁₋₈alkyl-NR⁵—CO-C₀₋₈alkyl,C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-CO—C₁₋₈alkyl-CO—NR⁵-C₀₋₈alkyl,C₀₋₈alkyl-O—C₂₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-O—C₀₋₈alkyl-CO—NR⁵-C₀₋₈alkyl,C₀₋₈alkyl-O-C₂₋₈alkyl-O—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-S—C₂₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-S(O_(n))-C₂₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-S—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-S(O_(n))-C₁₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl-S—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—CO—C₁₋₈alkyl-S(O_(n))—C₀₋₈alkyl,C₀₋₈alkyl-CO—NR⁵-C₂₋₈alkyl-S—C₀₋₈alkyl,C₀₋₈alkyl-CO-NR⁵-C₂₋₈alkyl-S(O_(n))-C₀₋₈alkyl,C₀₋₈alkyl-NR⁵-C₀₋₈alkyl-CO₂—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵-C₀₋₈alkyl-CS—O—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵-C₀₋₈alkyl-CO—NR—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—C₀₋₈alkyl-CS—NR⁵-C₀₋₈alkyl,C₀₋₈alkyl-O—C₀₋₈alkyl-CO₂—C₀₋₈alkyl, C₀₋₈alkyl-O—C₀₋₈alkyl-CS—C₀₋₈alkyl;C₀₋₈alkyl-SiR⁷R⁸-C₀₋₈alkyl, C₀₋₈alkyl-SiR⁷R⁸-C₀₋₈alkyl-NR⁶—CO—C₀₋₈alkyland C₀₋₈alkyl-SiR⁷R⁸-C₀₋₈alkyl-CO—NR⁶—C₀₋₈alkyl.

[0061] More preferred “A” substituents are selected from the groupconsisting of C₀₋₈alkyl-CO—NR⁵-C₀₋₈alkyl,C₀₋₈alkyl-O—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-O—C₂₋₈alkyl-O-C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-S—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-S(O_(n))-C₁₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl-S—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—CO-C₁₋₈alkyl-S(O_(n))—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—C₀₋₈alkyl-CO₂—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-O—C₀₋₈alkyl-CO₂-C₀₋₈alkyl.

[0062] Preferably, “Z” is —NH—C(NR⁹R¹⁰)═NR. Preferably R⁹, R¹⁰ and R¹¹are independently selected from the group consisting of H and C₁₋₆alkyl.More preferably, R⁹, R¹⁰ and R¹¹ are H.

[0063] Preferred R² substituents are —SO₂-aryl and —SO₂-C₁₋₁₀alkyl. Morepreferably, R² is —SO₂-aryl.

[0064] Preferred R³ substituents are H and C₁₋₈alkyl. More preferably,R³ is H.

[0065] Preferred compounds and subgroups of compounds may be selectedfrom any combination of the formulas presented in this specificationwith one or more of the preferred groupings of substituents at aparticular location.

[0066] Other preferred compounds of the present invention are shown butnot limited to the following list of compounds which have the structure:

[0067] This invention also encompasses prodrug derivatives of thecompounds contained herein. The term “prodrug” refers to apharmacologically-inactive derivative of a parent drug molecule thatrequires biotransformation, either spontaneous or enzymatic, within theorganism to release the active drug. Prodrugs are variations orderivatives of the compounds of this invention which have metabolicallycleavable groups and become, by solvolysis under physiologicalconditions, or by enzymatic degradation, compounds which arepharmaceutically active in vivo. Prodrug compounds of this invention maybe called single, double, triple etc., depending on the number ofbiotransformation steps required to release the active drug within theorganism, and indicating the number of functionalities present in aprecursor-type form. Prodrug forms often offer advantages of solubility,tissue compatibility, or delayed release in the mammalian organism (see,Bundgard, H., Design of Prodruqs, pp. 7-9, 21-24, Elsevier, Amsterdam1985 and Silverman, R. B., The Organic Chemistry of Drug Design and DrugAction, pp. 352-401, Academic Press, San Diego, Calif., 1992). Prodrugscommonly known in the art include acid derivatives well known topractitioners of the art, such as, for example, esters prepared byreaction of the parent acids with a suitable alcohol, or amides preparedby reaction of the parent acid compound with an amine, or basic groupsreacted to form an acylated base derivative. Moreover, the prodrugderivatives of this invention may be combined with other features hereintaught to enhance bioavailability.

[0068] Preparation of Compounds

[0069] The compounds of the present invention may be synthesized byeither solid or liquid phase methods described and referenced instandard textbooks, or by a combination of both methods. These methodsare well known in the art. See, Bodanszky, M., in “The Principles ofPeptide Synthesis”, Hafner, K., Rees, C. W., Trost, B. M., Lehn, J.-M.,Schleyer, P. v-R., Zahradnik, R., Eds., Springer-Verlag, Berlin, 1984.Starting materials are commercially available reagents and reactions arecarried out in standard laboratory glassware and reaction vessels underreaction conditions of standard temperature and pressure, except whereotherwise indicated.

[0070] The starting materials used in any of these methods arecommercially available from chemical vendors such as Aldrich, Sigma,Nova Biochemicals, Bachem Biosciences, and the like, or may be readilysynthesized by known procedures.

[0071] During the synthesis of these compounds, the functional groups ofthe amino acid derivatives used in these methods are protected byblocking groups to prevent cross reaction during the coupling procedure.Examples of suitable blocking groups and their use are described in “ThePeptides: Analysis, Synthesis, Biology”, Academic Press, Vol. 3 (Gross,E. & Meienhofer, J., Eds., 1981) and Vol. 9 (, S. &., Eds., 1987), thedisclosures of which are incorporated herein by reference.

[0072] Nine exemplary synthesis schemes are outlined below, and thespecific syntheses are described in the Examples. The reaction productsare isolated and purified by conventional methods, typically by solventextraction into a compatible solvent. The products may be furtherpurified by column chromatography or other appropriate methods.

[0073] The preparation of the carbamate containing compound 107 typifiesthe construction of compounds containing this linkage (Scheme 3-A). Inthe first step, a suitable protected amino alcohol (illustrated withanalog 100) is allowed to react with excess phosgene to provide theintermediate chloroformate (101). This material is condensed with thedifferentially protected diamino propionate derivative 103, thusproducing the carbamate linked intermediate (104). The CBz group on theterminal amine is then removed with palladium and hydrogen (105) and theformed amine is reacted withN,N′-bis(tert-butoxycarbonyl)-S-methoxy-isothiourea producing theprotected guanidine derivative (106). The compound is completelydeprotected with neat TFA at room temperature and then subjected to asalt exchange with HCI providing the desired amino acids (107).Compounds containing a urea linkage (113) are prepared in an analogousmanner starting from differentially protected diamines (Scheme 3-B).

[0074] Compounds that contain an amine linkage (118) can be prepared ina similar fashion (Scheme 3-A). Protected bromo-amine 114 (prepared fromalcohol 113 by treatment with CBr₄ and Ph₃P) is allowed to react withamine 103 in the presence of K₂CO₃ which forms adduct 115. Subjection ofprotected derivative 115 to the same sequence of reactions outlined forthe transformation of 104 into 107 affords the desired amine containingcompound 118.

[0075] Compositions and Formulations

[0076] The compounds of this invention may be isolated as the free acidor base or converted to salts of various inorganic and organic acids andbases. Such salts are within the scope of this invention. Non-toxic andphysiologically-compatible salts are particularly useful although otherless desirable salts may have use in the processes of isolation andpurification.

[0077] A number of methods are useful for the preparation of the saltsdescribed above and are known to those skilled in the art. For example,reaction of the free acid or free base form of a compound of thestructures recited above with one or more molar equivalents of thedesired acid or base in a solvent or solvent mixture in which the saltis insoluble, or in a solvent like water after which the solvent isremoved by evaporation, distillation or freeze drying. Alternatively,the free acid or base form of the product may be passed over an ionexchange resin to form the desired salt or one salt form of the productmay be converted to another using the same general process.

[0078] Diagnostic applications of the compounds of this invention willtypically utilize formulations such as solution or suspension. In themanagement of thrombotic disorders the compounds of this invention maybe utilized in compositions such as tablets, capsules or elixirs fororal administration, suppositories, sterile solutions or suspensions orinjectable administration, and the like, or incorporated into shapedarticles. Subjects in need of treatment (typically mammalian) using thecompounds of this invention can be administered dosages that willprovide optimal efficacy. The dose and method of administration willvary from subject to subject and be dependent upon such factors as thetype of mammal being treated, its sex, weight, diet, concurrentmedication, overall clinical condition, the particular compoundsemployed, the specific use for which these compounds are employed, andother factors which those skilled in the medical arts will recognize.

[0079] Formulations of the compounds of this invention are prepared forstorage or administration by mixing the compound having a desired degreeof purity with physiologically acceptable carriers, excipients,stabilizers etc., and may be provided in sustained release or timedrelease formulations. Acceptable carriers or diluents for therapeuticuse are well known in the pharmaceutical field, and are described, forexample, in Remington's Pharmaceutical Sciences, Mack Publishing Co.,(A. R. Gennaro edit. 1985). Such materials are nontoxic to therecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, acetate and other organic acidsalts, antioxidants such as ascorbic acid, low molecular weight (lessthan about ten residues) peptides such as polyarginine, proteins, suchas serum albumin, gelatin, or immunoglobulins, hydrophilic polymers suchas polyvinylpyrrolidinone, amino acids such as glycine, glutamic acid,aspartic acid, or arginine, monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannoseor dextrins, chelating agents such as EDTA, sugar alcohols such asmannitol or sorbitol, counterions such as sodium and/or nonionicsurfactants such as Tween, Pluronics or polyethyleneglycol.

[0080] Dosage formulations of the compounds of this invention to be usedfor therapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile membranes such as 0.2 micronmembranes, or by other conventional methods. Formulations typically willbe stored in lyophilized form or as an aqueous solution. The pH of thepreparations of this invention typically will be between 3 and 11, morepreferably from 5 to 9 and most preferably from 7 to 8. It will beunderstood that use of certain of the foregoing excipients, carriers, orstabilizers will result in the formation of cyclic polypeptide salts.While the preferred route of administration is by injection, othermethods of administration are also anticipated such as intravenously(bolus and/or infusion), subcutaneously, intramuscularly, colonically,rectally, nasally or intraperitoneally, employing a variety of dosageforms such as suppositories, implanted pellets or small cylinders,aerosols, oral dosage formulations and topical formulations such asointments, drops and dermal patches. The compounds of this invention aredesirably incorporated into shaped articles such as implants which mayemploy inert materials such as biodegradable polymers or syntheticsilicones, for example, Silastic, silicone rubber or other polymerscommercially available.

[0081] The compounds of this invention may also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of lipids, such as cholesterol, stearylamine orphosphatidylcholines.

[0082] The compounds of this invention may also be delivered by the useof antibodies, antibody fragments, growth factors, hormones, or othertargeting moieties, to which the compound molecules are coupled. Thecompounds of this invention may also be coupled with suitable polymersas targetable drug carriers. Such polymers can includepolyvinylpyrrolidinone, pyran copolymer,polyhydroxy-propyl-methacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, inhibitors of thisinvention may be coupled to a class of biodegradable polymers useful inachieving controlled release of a drug, for example polylactic acid,polyglycolic acid, copolymers of polylactic and polyglycolic acid,polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,polyacetals, polydihydropyrans, polycyanoacrylates and cross linked oramphipathic block copolymers of hydrogels. Polymers and semipermeablepolymer matrices may be formed into shaped articles, such as valves,stents, tubing, prostheses and the like.

[0083] Therapeutic compound liquid formulations generally are placedinto a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable byhypodermic injection needle.

[0084] Therapeutically-effective dosages may be determined by either invitro or in vivo methods. For each particular compound of the presentinvention, individual determinations may be made to determine theoptimal dosage required. The range of therapeutically-effective dosageswill naturally be influenced by the route of administration, thetherapeutic objectives, and the condition of the patient. For injectionby hypodermic needle, it may be assumed the dosage is delivered into thebodys fluids. For other routes of administration, the absorptionefficiency must be individually determined for each inhibitor by methodswell known in pharmacology. Accordingly, it may be necessary for thetherapist to titer the dosage and modify the route of administration asrequired to obtain the optimal therapeutic effect. The determination ofeffective dosage levels, that is, the dosage levels necessary to achievethe desired result, will be within the ambit of one skilled in the art.Typically, applications of compound are commenced at lower dosagelevels, with dosage levels being increased until the desired effect isachieved.

[0085] A typical dosage might range from about 0.001 mg/kg to about 1000mg/kg, preferably from about 0.01 mg/kg to about 100 mg/kg, and morepreferably from about 0.10 mg/kg to about 20 mg/kg. Advantageously, thecompounds of this invention may be administered several times daily, andother dosage regimens may also be useful.

[0086] Typically, about 0.5 to 500 mg of a compound or mixture ofcompounds of this invention, as the free acid or base form or as apharmaceutically-acceptable salt, is compounded with a physiologicallyacceptable vehicle, carrier, excipient, binder, preservative,stabilizer, dye, flavor etc., as called for by accepted pharmaceuticalpractice. The amount of active ingredient in these compositions is suchthat a suitable dosage in the range indicated is obtained.

[0087] Typical adjuvants which may be incorporated into tablets,capsules and the like are a binder such as acacia, corn starch orgelatin, and excipient such as microcrystalline cellulose, adisintegrating agent like corn starch or alginic acid, a lubricant suchas magnesium stearate, a sweetening agent such as sucrose or lactose, ora flavoring agent. When a dosage form is a capsule, in addition to theabove materials it may also contain a liquid carrier such as water,saline, or a fatty oil. Other materials of various types may be used ascoatings or as modifiers of the physical form of the dosage unit.Sterile compositions for injection can be formulated according toconventional pharmaceutical practice. For example, dissolution orsuspension of the active compound in a vehicle such as an oil or asynthetic fatty vehicle like ethyl oleate, or into a liposome may bedesired. Buffers, preservatives, antioxidants and the like can beincorporated according to accepted pharmaceutical practice.

[0088] In practicing the methods of this invention, the compounds ofthis invention may be used alone, or in combination with othertherapeutic or diagnostic agents. In certain preferred embodiments, thecompounds of this invention may be coadministered along with othercompounds typically prescribed for these conditions according togenerally accepted medical practice, such as anticoagulant agents,thrombolytic agents, or other antithrombotics, including plateletaggregation inhibitors, tissue plasminogen activators, urokinase,prourokinase, streptokinase, heparin, aspirin, or warfarin. Thecompounds of this invention can be utilized in vivo, ordinarily inmammals such as primates, (e.g. humans), sheep, horses, cattle, pigs,dogs, cats, rats and mice, or in vitro.

[0089] With respect to the coronary arterial vasculature, abnormalthrombus formation characterizes the rupture of an establishedatherosclerotic plaque which is the major cause of acute myocardialinfarction and unstable angina, as well as also characterizing theocclusive coronary thrombus formation resulting from either thrombolytictherapy or percutaneous transluminal coronary angioplasty (PTCA).

[0090] The compounds of the present invention preferably have an IC₅₀ ofless than about 2.0 μM, more preferably less than about 1.0 μM, and mostpreferably less than about 200 nM, as measured by one or more of theassays described herein. These compounds, selected and used as disclosedherein, are believed to be useful for preventing or treating a conditioncharacterized by undesired thrombosis, such as, by way of illustrationand not limitation, (a) the treatment or prevention of anythrombotically mediated acute coronary syndrome including myocardialinfarction, unstable angina, refractory angina, occlusive coronarythrombus occurring post-thrombolytic therapy or post-coronaryangioplasty, (b) the treatment or prevention of any thromboticallymediated cerebrovascular syndrome including embolic stroke, thromboticstroke or transient ischemic attacks, (c) the treatment or prevention ofany thrombotic syndrome occurring in the venous system including deepvenous thrombosis or pulmonary embolus occurring either spontaneously orin the setting of malignancy, surgery or trauma, thromboticthrombocytopenic purpura, thromboangiitis obliterans, or thromboticdisease associated with heparin induced thrombocytopenia, (e) thetreatment or prevention of thrombotic complications associated withextracorporeal circulation (e.g. renal dialysis, cardiopulmonary bypassor other oxygenation procedure, plasmapheresis), (f) coagulopathy anddisseminated intravascular coagulation (g) the treatment or preventionof thrombotic complications associated with instrumentation (e.g.cardiac or other intravascular catheterization, intra-aortic balloonpump, coronary stent or cardiac valve), (h) those involved with thefitting of prosthetic devices, (i) vascularization of solid tumors and(j) retinopathy.

[0091] Without further description, it is believed that one of ordinaryskill in the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out preferred embodiments of thepresent invention, and are not to be construed as limiting in any waythe remainder of the disclosure.

EXAMPLES Example 1

[0092]

[0093] Part A: Synthesis of2-(n-Butylsulfonylamido)-3-(N-Boc)-aminopropionic acid hydroxymethylresin (1)

[0094] Hydroxymethyl resin (2.7 g, 2.1 mmol, 0.8 mmol/ g) was suspendedin DMF (20 ml). 2-(n-Butylsulfonylamido)-3-(N-Boc)-aminopropionic acid (prepared according to Claremon, D. A., et al., PCT/US94/01881) (1.0 g,3.0 mmol), BOP (2.8 g, 6.3 mmol), DIEA (2.0 ml, 11.1 mmol) and catalyticamount of DMAP were added. The mixture was rotated on a nutator at r.t.overnight. The resulting resin (1) was collected in a fritted glassBuchner funnel, washed with MeOH, CH₂Cl₂ and dried in vacuo.

[0095] Part B:

[0096] Compound (2) was synthesized by standard automated solid-phasesynthesis protocols using an Applied Biosystems 431 A PeptideSynthesizer. The resin (1) was deprotected with 50% TFA/CH₂Cl₂ followedby coupling with N-Boc-β-alanine. The Boc group was removed again with50% TFA/CH₂Cl₂ followed by coupling with N-Boc-glycine. The Boc groupwas removed with TFA and the resin dried.

[0097] Part C:

[0098] The resin (2) (0.5 mmol of peptide theoretical) was neutralizedwith 10% DIEA/CH₂Cl₂ and swelled and saturated with DMF (15 ml).1H-Pyrazole-1-carboxamidine hydrochloride (2 g, 13.8 mmol) and DIEA (2.9ml, 15.7 mmol) were added to the resin mixture. The reaction was allowedto proceed at 37° for 2 hr. after which the Kaiser ninhydrin test of aresin sample was negative. The resulting resin was washed with CH₂Cl₂and CH₃OH and dried in vacuo.

[0099] Part D: HF Cleavage of the resin (3).

[0100] The resin (3) (1 g) was suspended in HF (10 ml/g resin)containing 10% by volume anisole and 2% methylethylsulfide (MES)employing a type 1B HF cleavage apparatus. The liquid HF was condensedinto the cleavage vessel with the aid of liquid nitrogen cooling and wasthen maintained at −10° C. for 30 min. and at 0° C. for an additional 30min. The HF was removed in vacuo and the resin transferred to a frittedglass funnel. The resin was washed with ether followed by extraction ofthe crude product from the resin with 2N aqueous acetic acid.Lyophilization of the extracts furnished a white powder. The product (4)was purified by preparative HPLC. MS (ES) 395 (M+H⁺)

Example 2

[0101]

[0102] Compound (5) was synthesized by the method of Example 1 usingN-Boc-β-alanine in place of N-Boc-glycine in Part B. MS (ES) 409 (M+H⁺)

Example 3

[0103]

[0104] Compound (6) was synthesized by the method of Example 1 usingN-Boc-4-aminobutyric acid in place of N-Boc-glycine in Part B. MS (ES)423 (M+H⁺)

Example 4

[0105]

[0106] Compound (7) was synthesized by the method of Example 1 usingN-Boc-5-aminovaleric acid in place of N-Boc-glycine in Part B. MS (ES)437 (M+H⁺)

Example 5

[0107]

[0108] Compound (8) was synthesized by the method of Example 1 using2-(benzenesulfonylamido)-3-(N-Boc)-aminopropionic acid (prepared asdescribed by Claremon, D. A., et al., PCT/US94/01881, the disclosure ofwhich is incorporated herein by reference) in place of2-(n-butylsulfonylamido)-3-(N-Boc)-aminopropionic acid. MS (ES) 443(M+H⁺)

Example 6

[0109]

[0110] PART A:

[0111] To the 2-(benzenesulfonylamido)-3-(N-Boc)-aminopropionic acidhydroxymethyl resin from Example 5 (0.75 g, 0.375 mmol) in a frittedreaction vessel, was added 40% TFA/CH₂Cl₂. The mixture was stirred atr.t. for 30 min. The resulting 2-(benzenesulfonylamido)-3-aminopropionicacid hydroxymethyl resin was collected through filtration, washed withCH₂Cl₂ and MeOH repeatedly and neutralized with 10% DIEA in CH₂Cl₂.Then, a solution of 8-(N-Boc) aminooctanoic acid (0.195 g, 0.75 mmol),BOP (0.5 g, 1.1 mmol) and DIEA (0.35 ml, 1.9 mmol) in 2 ml of DMF wasadded to the neutralized resin. The reaction proceeded on a nutator atr.t. for 8 hr., after which the Kaiser ninhydrin test of a resin samplewas negative. The mixture was filtered and the resin collected. Theresin was washed with CH₂Cl₂, MeOH, dried in vacuo and the Boc groupcleaved with 40% TFA/CH₂Cl₂. The resulting free amino-containing resinwas washed and dried in vacuo to yield titled compound (10).

[0112] Part B:

[0113] Compound (9) was synthesized by the method of Part C and D ofExample 1 using (10) in place of (2) in Part B. MS (ES) 428 (M+H⁺)

Example 7

[0114]

[0115] Compound (11) was synthesized by the method of Example 6 usingp-toluenesulfonyl chloride in place of benzenesulfonyl chloride. MS (ES)442 (M+H⁺)

Example 8

[0116]

[0117] Compound (12) was synthesized by the method of Example 6 usingα-toluenesulfonyl chloride in place of benzenesulfonyl chloride. MS (ES)442 (M+H⁺)

Example 9

[0118]

[0119] Compound (13) was synthesized by the method of Part B, C, and Dof Example 1 using N-Boc-β-alanine, N-Boc-isonipecotic acid and2-(benzenesulfonylamido)-3-(N-Boc)-aminopropionic acid hydroxymethylresin. MS (ES) 469 (M+H⁺)

Example 10

[0120]

[0121] Compound (14) was synthesized by the method of Example 9 usingN-Boc-4-aminobutyric acid in place of N-Boc-β-alanine. MS (ES) 483(M+H⁺)

Example 11

[0122]

[0123] Compound (15) was synthesized by the method of Example 9 usingN-Boc-5-aminovaleric acid in place of N-Boc-β-alanine. MS (ES) 497(M+H⁺)

Example 12

[0124]

[0125] Compound (16) was synthesized by the method of Example 9(omitting the step of guanylating the nitrogen) using N-Boc-isonipecoticacid in place of N-Boc-β-alanine at the N-terminus and N-Boc-glycine inplace of N-Boc-isonipecotic acid as the central spacer. MS (ES) 413(M+H⁺)

Example 13

[0126]

[0127] Compound (17) was synthesized by the method of Example 12 usingN-Boc-β-alanine in place of N-Boc-glycine. MS (ES) 427 (M+H⁺)

Example 14

[0128]

[0129] Compound (18) was synthesized by the method of Example 12 usingN-Boc-nipecotic acid in place of N-Boc-isonipecotic acid. MS (ES) 413(M+H⁺)

Example 15

[0130]

[0131] Compound (19) was synthesized by the method of Part A of Example6 using trans-(Boc-4-aminomethyl)-cyclohexanecarboxylic acid andN-Boc-4-aminobutyric acid in place of 8-(N-Boc)aminooctanoic acid andusing HBTU in place of BOP.

[0132] Compound (20) was synthesized from compound (19) by the method ofpart C and D of Example 1. MS (ES) 511 (M+H)⁺

Example 16

[0133]

[0134] Preparation of 107 (Scheme 3-A)

[0135] Step 1.

[0136] A mixture of CBz protected amino-hexanol 100 (0.10 g, 0.39 mmol)was dissolved in toluene (5 mL) and treated with a 2M solution ofphosgene in toluene (2 mL). This solution was allowed to stir for 2 h atroom temperature and was then concentrated to dryness. The residue wasthen dissolved in CH₂Cl₂ (5 mL) and added to a solution of 103 (0.0.12g, 0.39 mmol), pyridine (1 mL) and CH₂Cl₂ (5 mL). The resulting solutionwas allowed to stir for 2 h. The solution was then diluted with EtOAc(100 mL) and washed with saturated NaHCO₃ and water. The organicmaterial was then dried (MgSO₄) an concentrated. The crude residue waspurified by chromatography (silica gel, Hexanes-EtOAc 1:1) giving 0.213g of 104.

[0137] Step 2.

[0138] A mixture of compound 104 (0.213 g), 10% palladium on carbon(0.2g), and ethanol (10 mL) was stirred under an atmosphere of hydrogenfor 2h and then filtered and concentrated providing 0.14 g of thedesired amine 105.

[0139] Step 3.

[0140] A mixture of the amine 105 (0.14 g, 0.32 mmol),N,N′-bis(tert-butoxycarbonyl)-S-methoxy-isothiourea (0.147 g, 0.49mmol), and CH₂Cl₂ (1 mL) was maintained at room temperature for 48 h andthen was concentrated. This material was then purified by chromatography(silica gel, hexanes-EtOAc - 1:1) providing 106.

[0141] Step 4.

[0142] A mixture of the 106 (0.1 g) was dissolved in anhydrous TFA (5mL) and maintained at room temperature for 1 h. This material was thenconcentrated to dryness and the resulting material was taken up in 2NHCl (5 mL) and lyophilized providing 107 as a white hygroscopic solid.MS (m/e) 430 (MH+).

Example 17

[0143]

[0144] Preparation of 113 (Scheme 3-B)

[0145] Mono CBz protected hexane diamine (108) was converted to the ureaderivative 110 following the same procedure employed for the preparationof 104 (Example 16 step 1). Compound 104 was further transformed byfirst removing the CBz protecting group using the procedure outlined inExample 16 step 2 which afforded amine 111. This material was convertedto the protected guanidine 112 using the procedure outlined in Example16 step 3. Finally the desired material (113) was obtained using theprocedure outlined in Example 16 step 4. MS (m/e) 428 (MH+).

Example 18

[0146]

[0147] Preparation of 118 (Scheme 3-C)

[0148] A mixture of the bromide 114 (0.11 g, 0.33 mmol), amine 103 (0.10g, 0.33 mmol), and K2CO3 (0.05 g, 0.33 mmol) in CH₃CN (1 mL) was stirredat 50° C. overnight. The mixture was then diluted with EtOAc (50 mL) andwashed with H₂O. The organic material was dried and concentrated.Chromatography (silica gel - EtOAc) gave 0.05 g of 115 as a clear oil.Compound 115 was further transformed by first removing the CBzprotecting group using the procedure outlined in Example 16 step 2 whichafforded amine 116. This material was converted to the protectedguanidine 117 using the procedure outlined in Example 16 step 3. Finallythe desired material (118) was obtained using the procedure outlined inExample 16 step 4. MS (m/e) 414 (MH+).

Example 19

[0149]

[0150] Preparation of 126 (Scheme 4)

[0151] 2-Phthaloylethoxy)ethanol 123 (200mg, 0.85mmol)[prepared understandard conditions from 2-(2-aminoethoxy)ethanol and phthalicanhydride] was treated with phosgene (2.2 mL, 1.93M in toluene, 4.3mmol). After 1h, the mixture was concentrated and the residue dissolvedin pyridine (2mL). This mixture was then added dropwise to a solution ofamine 103 (255 mg, 0.85 mmol) in pyridine (2 mL). After 1 h, the mixturewas poured into H₂O and ethyl acetate (EtOAc). The layers wereseparated, the aqueous layer washed with EtOAc (2×), and the combinedextracts were dried (MgSO₄) and concentrated. The residue was purifiedby chromatography (SiO₂, 1:1 hexane:EtOAc) affording 180 mg (32%) of thedesired carbamate. Deprotection of the phthalimide was accomplished bytreatment of the carbamate product with hydrazine hydrate (0.80 mL) inethanol (2 mL). After 1 h, the mixture was concentrated, partitionedbetween NaHCO₃(aq) and EtOAc, and the layers were separated. The organicextract was washed with NaHCO₃(aq)(2×), dried (MgSO₄), and concentratedyielding 66 mg (48%) of the corresponding amine. The protected guanidinewas prepared according to a similar procedure described for thepreparation of 107 (Scheme 1). Deprotection with trifluoroacetic acid(TFA) yielded the TFA salt of the desired product 126. MS (m/e) 418(MH+).

Example 20

[0152]

[0153] Preparation of 127 (Scheme 4)

[0154] Step 1.

[0155] A solution of CBz protected 3-aminopropanol 119 (500 mg, 0.24mmol) and rhodium(II) acetate dimer (10 mg) in CH₂Cl₂ (40 mL) wastreated dropwise with a CH₂ Cl₂ solution of ethyl diazoacetate (0.28 mL,2.63 mmol). After 1 h, the mixture was concentrated and the residue waspurified by flash chromatography (SiO₂, 1:1 hexane:EtOAc) affording 560mg (79 %) of the ethyl ester. The ester (300 mg) was dissolved in EtOH(10 mL) and treated with NaOH (203 mg, 0.51 mmol). After 1 h, themixture was concentrated and the residue dissolved in H₂O. The pH wasadjusted to -2-3 using 1N HCl and then EtOAc was added and the layerswere separated. The aqueous layer was washed with EtOAc (2×), thecombined extracts were then dried (MgSO₄) and concentrated yielding 227mg (83%) of acid 121.

[0156] Step 2.

[0157] A solution of acid 121 (227 mg, 0.085 mmol) and N-methylmorpholine (0.94 mL, 0.085 mmol) in THF (4.5 mL) at -10 C was treatedwith ethyl chloroformate (0.82 mL, 0.085 mmol). After 0.25 h, themixture was treated with sodium borohydride (96 mg, 0.26 mmol) followedby dropwise addition of methanol (9 mL). The mixture was allowed towarm, treated with 10% acetic acid (aq), and concentrated. The residuewas partitioned between 1N NaOH and EtOAc, the layers were seperated,the aqueous was washed with EtOAc (2 x) and the combined extracts weredried (MgSO4) and concentrated. The residue was purified bychromatography (SiO2, 65:35 EtOAc:hexane) yielding 75 mg (35%) ofalcohol 124.

[0158] Step 3.

[0159] The carbamate derivative 127 was prepared following the sameprocedures described for the preparation of 107 (Scheme 1). Alcohol 124(75 mg) was treated with phosgene followed by amine 103 (89 mg) yieldingthe carbamate (63 mg). Deprotection of the CBz by hydrogenolysis andsubsequent treatment of the resulting amine (50 mg) withN,N′-bis(tert-butoxycarbonyl)-S-methoxy-isothiourea (35 mg) yielded theprotected guanidine (30 mg). Deprotection with trifluoroacetic acidyielded the TFA salt of the desired product 127. MS (m/e) 432 (MH+).

Example 21

[0160]

[0161] Preparation of 128 (Scheme 4)

[0162] The product 128 was prepared following the same generalprocedures described for the preparation of 127. CBz protected4-aminobutanol (1.0 g) was treated with rhodium(II) acetate dimer (10mg) and ethyl diazoacetate (0.52 mL) yielding the homologated ester (440mg). Saponification of the ester (728 mg) with NaOH (470 mg) andreduction of the resulting acid 122 (590 mg) with ethyl chloroformate(0.20 mL), N-methyl morpholine (0.23 mL) and sodium borohydride (238 mg)yielded alcohol 125 (134 mg). Treatment of 125 with phosgene followed byamine 103 (151 mg) yielded the desired carbamate (127 mg). Deprotectionof the CBz by hydrogenolysis and subsequent treatment of the resultingamine (42 mg) with N,N′-bis(tert-butoxycarbonyl)-S-methoxy-isothiourea(30 mg) yielded the protected guanidine (23 mg). Deprotection withtrifluoroacetic acid yielded the TFA salt of the desired product 128. MS(m/e) 446 (MH+).

Example 22

[0163]

[0164] Preparation of 131 (Scheme 5)

[0165] Step 1.

[0166] A solution of oxalyl chloride (0.99 mL, 11.3 mmol) in CH2Cl2 (40mL) at −78 C. was treated with dimethyl sulfoxide (0.86 mL, 12.2 mmol).After 0.5 h, a solution of alcohol 129 (1.94 g, 8.12 mmol) in CH2Cl2 (7mL) was added dropwise. After 0.5 h, triethylamine (2.05 g, 20.3 mmol)was added and the cooling bath was removed. After 1 h, the mixture waspoured into EtOAc and NH₄Cl(aq). The layers were separated, the aqueouslayer was washed with EtOAc and the combined extracts were dried(MgSO₄). Concentration yielded the corresponding aldehyde. A solution ofthis crude aldehyde in THF (20 mL) was then treated with(carbethoxymethylene)triphenylphosphorane (2.96 g, 8.50 mmol). After 62h, the mixture was concentrated and the residue purified bychromatography (SiO₂, 4:1 to 3:2 hexane:EtOAc) yielding the acrylate asa mixture of isomers. A sample of the trans isomer (490 mg, 1.60 mmol)in THF (5 mL) at −78 ° C. was treated with diisobutylaluminum hydride(Dibal-H) (5 mL, 1.0M in toluene, 5.0 mmol). After 1.5 h, the mixturewas treated with EtOAc (5 mL) followed by a saturated aqueous solutionof NH₄Cl (0.4 mL). After 1 h, the mixture was treated with SiO₂ gel (˜4mL), diluted with EtOAc, and filtered through MgSO₄ and Celite.Concentration yielded 237 mg (56%) of the alcohol 130.

[0167] Step 2.

[0168] The carbamate derivative 131 was prepared following the sameprocedures described for the preparation of 107 (Scheme 1). Alcohol 130(237 mg) was treated with phosgene followed by amine 103 (263 mg)yielding the carbamate (368 mg). Deprotection of the CBz byhydrogenolysis and subsequent treatment of the resulting amine (116 mg)with N,N′-bis(tert-butoxycarbonyl)-S-methoxy-isothiourea (147 mg)yielded the protected guanidine (104 mg, 59%). Deprotection withtrifluoroacetic acid yielded the TFA salt which upon treatment withaqueous HCl and lyophilization yielded the HCl salt of the desiredproduct 131. MS (m/e) 446 (M+).

Example 23

[0169]

[0170] Preparation of 137 (Scheme 6)

[0171] Step 1.

[0172] A solution of acid 121 (418 mg, 1.56 mmol) and amine 103 (470 mg,1.56 mmol) in CH₂Cl₂ (8 mL) was treated with1-[3-(dimethylamino)propyl]-3-ethylcarbodiimde hydrochloride (EDCl) (300mg, 1.56 mmol) and triethylamine (0.55 mL, 3.9 mmol). After 2 h, themixture was concentrated and the residue purified by chromatography(SiO₂, 4:1 EtOAc:hexane) affording 60 mg (7%) of the amide 134.

[0173] Step 2.

[0174] The amide derivative 137 was then prepared following the sameprocedures described for 107 (Scheme 3-A, steps 2-4). Hydrogenolysis of134 (152 mg) yielded the corresponding amine (130 mg). Treatment of theamine (111 mg) with N,N-bis(tert-butoxycarbonyl)-S-methoxy-isothiourea(85 mg) yielded the protected guanidine (100 mg, 57%). Deprotection withtrifluoroacetic acid yielded the TFA salt which upon treatment withaqueous HCl and lyophilization yielded the HCl salt of the desiredproduct 137. MS (m/e) 446 (M+).

Example 24

[0175]

[0176] Preparation of 138 (Scheme 6)

[0177] The amide derivative 138 was prepared following the same generalprocedures described for the preparation of 137 (Scheme 6). Treatment ofacid 132 (128 mg, prepared according to the general procedure describedfor 121, Scheme 4 starting from CBz protected 5-aminopentanol) withamine 103 (130 mg) and EDCl (83 mg) in CH₂Cl₂ yielded amide 135 (140 mg,56%). Hydrogenolysis of 135 (310 mg) yielded the corresponding amine(226 mg). Treatment of the amine (210 mg) withN,N′-bis(tert-butoxycarbonyl)-S-methoxy-isothiourea (155 mg) yielded theprotected guanidine (160 mg, 49%). Deprotection with trifluoroaceticacid yielded the TFA salt which upon treatment with aqueous HCl andlyophilization yielded the HCl salt of the desired product 138. MS (m/e)430 (M+).

Example 25

[0178]

[0179] Preparation of 139 (Scheme 6)

[0180] The amide derivative 139 was prepared following the same generalprocedures described for the preparation of 137 (Scheme 6). Treatment ofacid 133 (200 mg, prepared according to the general procedure describedfor 121, Scheme 4 starting from CBz protected 6-aminohexanol) with amine103 (195 mg), EDCl (124 mg), and triethylamine (0.23 mL) in CH₂ Cl₂yielded amide 136 (169 mg, 44%). Hydrogenolysis of 136 (294 mg) yieldedthe corresponding amine (141 mg). Treatment of the amine (120 mg) withN,N′-bis(tert-butoxycarbonyl)-S-methoxy-isothiourea (84 mg) yielded theprotected guanidine (55 mg, 30%). Deprotection with trifluoroacetic acidyielded the TFA salt which upon treatment with aqueous HCl andlyophilization yielded the HCl salt of the desired product 139. MS (m/e)444 (M+).

Example 26

[0181]

[0182] Preparation of 140 (Scheme 6)

[0183] The amide derivative 149 was prepared following the same generalprocedures described for the preparation of acid 121 (Scheme 4) andamide 137 (Scheme 6). Alcohol 129 (1.45 g, 6.07 mmol) was treated withethyl diazoacetate (0.70 mL, 6.68 mmol) and rhodium(II) acetate dimer(75 mg) yielding the corresponding ester (993 mg, 50%). Treatment of theester (525 mg) with sodium hydroxide (324 mg) in EtOH afforded the acidwhich was then treated with amine 103 (450 mg) and EDCl (311 mg) inCH₂Cl₂ (5 mL) yielding the amide (865 mg, 96%). Hydrogenolysis of theamide (865 mg) yielded the corresponding primary amine (583 mg).Treatment of the amine (355 mg) withN,N′-bis(tert-butoxycarbonyl)-S-methoxy-isothiourea (441 mg) yielded theprotected guanidine (68 mg, 11%). Deprotection with trifluoroacetic acidyielded the TFA salt which upon treatment with aqueous HCl andlyophilization yielded the HCl salt of the desired product 139. MS (m/e)432 (M+).

Example 27

[0184]

[0185] Preparation of 145 (Scheme 7)

[0186] Step 1.

[0187] A solution of 141 (5.0 g, 26 mmol) in 1:1 H20: dioxane (32 mL) at0 C. was treated with sodium hydroxide (2.14 g, 53 mmol) followed bybenzenesulfonyl chloride (3.8 mL, 29 mmol). Upon consumption of 141, themixture was concentrated and the pH of the mixture was adjusted to ˜4 byaddition of 1N HCl. The aqueous layer was washed with EtOAc (3×) and thecombined extracts were dried (MgSO₄) and concentrated yielding the crudesulfonamide 142 which was used without further purification.

[0188] Step 2.

[0189] A solution of 1,7-diaminoheptane 143 (3.0 g, 23 mmol) in DMF (10mL) was treated with N,N′-bis(tert-butoxycarbonyl)-S-methoxy-isothiourea (3.3 g, 12 mmol). Themixture was diluted with H₂O and EtOAc and the layers separated. Theaqueous layer was washed with EtOAc (3×) and the combined extracts werewashed with 10% citric acid (aq). The aqueous layer was then treatedwith potassium carbonate until a pH of ˜10 was achieved and then washedwith EtOAc (3×). The combined extracts were dried (K₂CO₃) andconcentrated to yield the crude amine 144 which was used without furtherpurification.

[0190] Step 3.

[0191] A solution of the crude acid 142 (72 mg) and amine 144 (123 mg)in CH₂Cl₂ was treated with EDCl (42 mg) and triethylamine (0.064 mL).After 16 h, the mixture was concentrated and the residue purified bychromatography (SiO₂, 4:1 to 1:1 hexane:EtOAc) yielding the desiredamide (39 mg). Deprotection of the amide with trifluoroacetic acidyielded the TFA salt of the desired product 145. MS (m/e) 428 (MH+).

Example 28

[0192] Assay Methods

[0193] The identification of compounds which are active plateletaggregation inhibitors is made possible by the observation thatcompounds which block the binding of fibrinogen to the GPIIb-IIIacomplex in vitro are also capable of inhibiting thrombin or. diphosphate(ADP)-induced aggregation of human platelets and the formation ofplatelet-thrombi in vivo. This observation provides the basis forobtaining potent platelet aggregation inhibitors by evaluating theability of test materials to disrupt fibrinogen-GPIIb-IIIa interactions.The ability of compounds to inhibit the adhesive function of otherintegrins that are closely related to GPIIb-IIIa can also be measured invitro by measuring their ability to inhibit the binding of adhesiveligands to various integrins such as vitronectin receptor (α_(v)β₃) andfibronectin receptor (α₅β₁).

[0194] The following assay methods were used to evaluate the compoundsof the invention.

[0195] Integrin Binding Assays

[0196] In the following assays, GPIIb-IIIa and vitronectin receptor,α_(v)β₃ were prepared in purified form, by methods described inFitzgerald, L. A., et al., Anal Biochem (1985) 151: 169-177 and Smith,J. W., J. Biol Chem (1988) 263: 18726-18731 (the disclosure of which isincorporated herein by reference). GPIIb-IIIa or vitronectin receptor,α_(v)β₃, are coated onto microtiter plates. The coated support is thencontacted with fibrinogen for the GPIIb-IIIa assay, or is contacted withvitronectin for the vitronectin receptor, α_(v)β₃ and with testmaterials and incubated for sufficient time to reach maximal ligandbinding to immobilized integrins. The adhesive ligands fibrinogen orvitronectin were typically provided at a concentration of 2-50 nM andthe test material can, if desired, be added at a series of diultions.Typical incubations were 2-4 hr at 25° C., the time and temperaturebeing interdependent.

[0197] Description of Purified Integrin Binding Assays

[0198] Purified platelet GPIIb-IIIa was prepared by Fitzgerald, L. A.,et al., Anal Biochem 151:169-177 (1985). Vitronectin receptor, α_(v)β₃,was prepared as described by Smith, J. W., J. Biol Chem (1988) 263:18726-18731. After purification, the receptors were each stored in 0.1%Triton X-100 at 0.1-1.0 mg/ml.

[0199] The receptors were coated to the wells of 96-well flat-bottomenzyme-linked immunoassay (ELISA) plates (Linbro EIA-Plus microtiterplate, Flow Laboratories) after diluting 1:200 with a solution of 20 mMTris-HCl, 150 mM NaCl, 1 mM CaCl₂, pH 7.4 to reduce the Triton X-100concentration to below its critical micellar concentration and adding analiquot of 100 μl to each well. The wells were allowed to incubateovernight at 4° C., and then aspirated to dryness. Additional sites wereblocked by the addition of bovine serum albumin (BSA) at 35 mg/ml in theabove buffer for 2 hr at 30° C. to prevent non-specific binding. Thewells were then washed once with binding buffer (50 nM Tris-HCl, 100 mMNaCl, 2 mM CaCl₂, 1 mg/ml BSA).

[0200] The corresponding ligands (fibrinogen, von Willebrand Factor, orvitronectin) were conjugated to biotin using commercially availablereagents and standard protocols. The labeled ligands were added to thereceptor-coated wells at a final concentration of 2-10 nM (100 μl/well)and incubated for 3 hr at 25° C. in the presence or absence of testsamples. After incubation, the wells were aspirated to dryness and boundligand containing biotin label was quantitated.

[0201] The bound protein was detected by the addition of anti-biotinantibody conjugated to alkaline phosphatase followed by addition ofsubstrate (p-nitrophenyl adenosine phosphate), and determination of theoptical density of each well at 405 nM. Decreased color development wasobserved in wells incubated with test samples which inhibited binding ofligand to receptor. From the various concentrations of test samples, theconcentration of inhibitor (IC₅₀) which half-maximally inhibits ligandbinding was determined

[0202] The Platelet Aggregation Assay

[0203] In addition to the ELISA integrin binding assays described above,the Aggregation-Human/PRP/ADP Assay is useful for evaluating therapeuticcompounds.

[0204] Platelet rich plasma (PRP) is prepared from healthy humanvolunteers for use in determining inhibition of platelet aggregation bycompounds. Blood was collected via a 21 gauge butterfly cannula, using atwo-syringe technique into {fraction (1/10)} volume of 105 trisodiumcitrate.

[0205] Platelet rich plasma was prepared at room temperature bycentrifugation of the citrated whole blood at 100×g for 15 minutes. ThePRP contained approximately 200-400,000 platelets/μl. Platelet poorplasma was prepared by centrifugation of citrated whole blood at12,000×g for 2 minutes.

[0206] Platelet aggregation was assayed in a Chrono-log whole bloodaggregometer (Chrono-log Corporation, Havertown, Pa.) using the PRPprepared above according to the manufacturers directions. Inhibition ofplatelet aggregation was studied by adding varying amounts of testmaterials followed by adenosine diphosphate (ADP, 20 μM) to stirredhuman PRP. Specifically, the human PRP was incubated with the compoundfor 1-2 min. at 37° C. prior to addition of the aggregating agent ADP.From full dose response curves the concentration (IC₅₀) tohalf-maximally inhibit platelet aggregation for test compounds wasdetermined. TABLE OF ASSAY TEST RESULTS GPIIbIIIa ELISA α_(v)β₃ ELISAPRP Compound # (IC₅₀ μM) (IC₅₀ μM) (IC₅₀ μM) 4 >100 >200 120 5 50 >10067.1 6 0.8 0.9 4.9 7 1 50 4.9 8 0.1 0.3 0.26 9 0.02 0.1 0.17 11 0.1 0.20.85 12 0.6 3.0 5.7 13 2.5 0.5 3.3 14 2.5 5.0 1.0 15 0.08 3.0 0.22 165.0 >100 49.3 17 0.1 >100 1.1 18 10 40 122 20 0.075 15 0.55

[0207] As can be seen from the compounds listed in the Table of AssayTest Results, a number of compounds of the invention display potentinhibition of ligand binding to GPIIb-IIIa and the vitronectin receptor,α_(v)β₃, as well as inhibition of ADP-induced human platelet aggregationwith IC₅₀'s which are submicromolar and would be expected to displayantiintegrin activities in vivo.

What is claimed is:
 1. A compound having the formula:

wherein: Y is selected from the group consisting of —COOH and —COOR⁴;where R⁴ is selected from the group consisting of C₁₋₁₀alkyl,C₁₋₈alkylaryl, and aryl-C₁₋₈alkyl; A is selected from the groupconsisting of C₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl, C₀₋₈alkyl-CO—NR⁵-C₀₋₈alkyl,C₀₋₈alkyl-NR⁵ —CO—C₁₋₈alkyl-NR⁵ —CO—C₀₋₈alkyl,C₀₋₈alkyl-CO—NR⁵—DC₁₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-CO—C₁₋₈aIkyl—CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl,C₀₋₈alkyl-O—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl, andC₀₋₈alkyl-O—C₂₋₈alkyl-O—C₀₋₈alkyl-CO—NR⁵—C₀₋₈alkyl; where R⁵ areindependently selected from the group consisting of H and C₁₋₆alkyl; Zis selected from the group consisting of —NH—C(NR⁹R¹⁰)═NR¹¹ and—C(NR⁹R¹⁰)═NR; where R⁹, R¹⁰ and R¹¹ are independently selected from thegroup consisting of H and C₁₋₆alkyl; R¹ is H; R² is selected from thegroup consisting of —SO_(m)-aryl and —SO_(m)—C₁₋₁₀alkyl, where m=2; andR³ is H; and all pharmaceutically-acceptable stereoisomers, salts,hydrates, solvates and prodrug derivatives thereof.
 2. The compound ofclaim 1 wherein Y is —COOH.
 3. The compound of claim 1 wherein R² is—SO₂-aryl.
 4. The compound of claim 1 wherein R⁴ is C₁₋₁₀alkyl.
 5. Thecompound of claim 1 wherein R⁹, R¹⁰ and R¹¹ are H.
 6. The compound ofclaim 1 wherein Z is —NH—C(NR⁹R¹⁰)═NR¹¹.
 7. The compound of claim 1wherein A is selected from the group consisting ofC₀₋₈alkyl-NR⁵—CO—C₀₋₈alkyl,C₀₋₈alkyl-NR⁵—CO—C₁₋₈alkyl-NR⁵—CO—C₀₋₈-alkyl,C₀₋₈alkyl-CO—C₁₋₈alkyl-CO—NR⁵—C₀₋₈alkyl, andC₀₋₈alkyl-O—C₀₋₈alkyl-CO—NR⁵-C₀₋₈alkyl.
 8. The compound of claim 7wherein Y is —COOH; Z is —NH— C(NR⁹R¹⁰)═NR¹¹; R⁹, R¹⁰ and R¹¹ are H; andR² is —SO₂-aryl.
 9. The compound of claim 1 wherein A is selected fromthe group consisting of C₀₋₈alkyl-NR⁵ —CO—C₁₋₈alkyl-NR⁵ —CO—C₀₋₈alkyl,and C₀₋₈alkyl-O—C₀₋₈alkyl-CO—NR⁵ —C₀₋₈alkyl.
 10. The compound of claim 9wherein Y is —COOH; Z is —NH—C(NR⁹R¹⁰)═NR¹¹; R⁹, R¹⁰ and R¹¹ are H; andR² is —SO₂-aryl.
 11. The compound of claim 1 , having an IC₅₀ of lessthan about 200 nM.
 12. A compound of claim 1 selected from the groupconsisting of:


13. A pharmaceutical composition for preventing or treating a conditionin a mammal characterized by undesired thrombosis comprising atherapeutically-acceptable carrier and a therapeutically-effectiveamount of a compound of claim 1 .
 14. A method for preventing ortreating a condition in a mammal characterized by undesired thrombosiscomprising administering to said mammal a therapeutically-effectiveamount of a compound of claim 1 .
 15. The method of claim 14 , whereinthe condition is selected from the group consisting of: acute coronarysyndrome, myocardial infarction, unstable angina, refractory angina,occlusive coronary thrombus occurring post-thrombolytic therapy orpost-coronary angioplasty, a thrombotically mediated cerebrovascularsyndrome, embolic stroke, thrombotic stroke, transient ischemic attacks,deep venous thrombosis, pulmonary embolus, coagulopathy, disseminatedintravascular coagulation, thrombotic thrombocytopenic purpura,thromboangiitis obliterans, thrombotic disease associated withheparin-induced thrombocytopenia, thrombotic complications associatedwith extracorporeal circulation, thrombotic complications associatedwith instrumentation such as cardiac or other intravascularcatheterization, intra-aortic balloon pump, coronary stent or cardiacvalve, conditions requiring the fitting of prosthetic devices,vascularization of solid tumors and retinopathy.
 16. A compound selectedfrom the group consisting of:


17. A compound selected from the group consisting of:

or a pharmaceutically acceptable stereoisomer, salt, solvate, hydrate,or prodrug derivative thereof.
 18. A compound of claim 17 , selectedfrom the group consisting of:


19. A compound of claim 17 , selected from the group consisting of:


20. A compound of claim 17 , selected from the group consisting of:


21. A pharmaceutical composition for preventing or treating a conditionin a mammal characterized by undesired thrombosis comprising atherapeutically-acceptable carrier and a therapeutically-effectiveamount of a compound of claim 17 .
 22. A method for preventing ortreating a condition in a mammal characterized by undesired thrombosiscomprising administering to said mammal a therapeutically-effectiveamount of a compound of claim 17 .
 23. The method of claim 22 , whereinthe condition is selected from the group consisting of: acute coronarysyndrome, myocardial infarction, unstable angina, refractory angina,occlusive coronary thrombus occurring post-thrombolytic therapy orpost-coronary angioplasty, a thrombotically mediated cerebrovascularsyndrome, embolic stroke, thrombotic stroke, transient ischemic attacks,deep venous thrombosis, pulmonary embolus, coagulopathy, disseminatedintravascular coagulation, thrombotic thrombocytopenic purpura,thromboangiitis obliterans, thrombotic disease associated withheparin-induced thrombocytopenia, thrombotic complications associatedwith extracorporeal circulation, thrombotic complications associatedwith instrumentation such as cardiac or other intravascularcatheterization, intra-aortic balloon pump, coronary stent or cardiacvalve, conditions requiring the fitting of prosthetic devices,vascularization of solid tumors and retinopathy.